The concept of multiplexing or “one sample application, multiple determinations” assay format had been first described by Tse Wen Chang in 1983 in his paper “Binding of cells to matrixes of distinct antibodies coated on solid surface,” J. Immunol. Methods 65 (1-2): 217-23. Multiplexing has the advantage of measuring multiple analytes, such as biological molecules like protein, DNA and RNA, in one reaction array, well or test tube. It reduces the size and amounts of samples and test reagents needed for the assays and achieves the goal of generating more data with reduced time and cost.
This multiplexing concept has been widely utilized in the post-genomic era. Companies like Agilent Technologies, Inc., Illumina, Inc., Affymetrix, Inc. and Life Technologies Corporation (ProtoArray®) have produced many high density multiplex array products that can test analytes ranging from hundreds to the whole genome. Recent progress has been made by companies like Luminex Corporation, NanoString Technologies, Inc., Sequenom Inc., Meso Scale Diagnostics, LLC (MSD) and many others that focus on the medium to low density multiplex assays. Multiplexing 1 to 40 analytes seems to have satisfied most of the practical needs for life science research or clinical diagnostics.
The crucial step in multiplexing is to associate each signal to its corresponding analyte. As an array is a systematic arrangement of objects, usually in rows and columns, computer software can easily track the position and the corresponding analyte. Consequently, Agilent Technologies, Inc., Illumina, Inc., Affymetrix, Inc., Life Technologies Corporation and many other companies specialized in high density multiplex array products rely on an array system to track signals to analytes [Tse Wen Chang, supra; Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. (1995), “Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray,” Science 270 (5235): 467-70]. Except MSD and others, companies focusing on the medium to low density multiplex assays like Luminex Corporation, NanoString Technologies, Inc., and Sequenom Inc. use different platforms from the array system to track analytes. These three appear to be the same in that they can multiplex in a well in suspension. However, they are quite different in how the signal is linked to its corresponding analyte. Luminex has its pride bead-based technology. Each bead or microsphere (about 5.6 microns) has a specific color code and serves as a barcode for a specific analyte. NanoString thrives on its digital molecular barcoding technology that tracks each analyte. It was invented by Dimitrov and Dunaway [“Direct multiplexed measurement of gene expression with color-coded probe pairs,” Nature Biotechnology 26 (3): 317-25 (2008)]. Sequenom identifies each analyte strictly by its mass. This technology is built on the speed and accuracy of Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS).
All the above multiplexing technologies have the following common aspects: They all depend on expensive and finicky equipment to detect signals and/or associate the signals to the identities of analytes. Reagents specific to each technology also need to be purchased by researchers. Basically, the essential lab equipment, such as a plate reader, in any life science or clinical laboratories cannot do the work. In addition, they are not efficient or flexible in testing a few samples or a few analytes within a day, which is usually desired in daily clinical practice. Furthermore, specially trained and dedicated technical staffs are required to operate those machines and interpret the raw data. Most of the labs either do not use multiplex assays or have to use core facilities to do the experiments. In the end, the advantages of multiplex assays, such as saving time or money, are not materialized at the end user level. Also, the current high or low density multiplex assays are so complex that it is difficult to generate reproducible data that can pass FDA regulations and be acceptable assays for clinical diagnostics.
Many companies have come up with solutions that do not require those high-tech machines. They are integrating the essential tools in all life science and clinical laboratories, such as microtiter plates, plate readers and qPCR machines. Those assays are singleplex assays measuring a few analytes on a 96-well plate or a 384-well plate. Major companies include Qiagen N.V. and Applied Biosystems/Life Technologies, Inc. Qiagen N.V. has produced many products to measure proteins or quantify nucleic acids, such as their Multi-Analyte ELISArray and RT2 Profiler PCR Array, that can work on routine lab equipment such as a qPCR machine or a plate reader. Essentially, the methods do enable the measurement of multiple analytes in one plate, and it also works well with small number of samples. However, they are singleplex, so they do not have the advantages of multiplex assays, such as reducing the size or quantity of samples or reagents and time and money. Applied Biosystems has also made many similar PCR arrays, which intrinsically have the same issues as Qiagen. Recently, Applied Biosystems did introduce an improved version of PCR arrays—the OpenArray. This platform does reduce the usage of samples and reagents and time with its 33 nl reaction volume and high throughput capacity. However, it runs into the same problems as the companies in the multiplexing arena—expensive, dedicated equipment and technical staff and is not efficient or flexible in testing a few samples or analytes and/or genes. So, these solutions do not keep the merits of multiplexing and address the issues of making multiplexing technologies readily available and simpler to use as essential lab tools.
Current target enrichment methods mainly use microspheres and/or centrifugation methods. “Protocol for the fast chromatin immunoprecipitation (CHIP) method” is a representative example of target enrichment approach [Joel D Nelson, Oleg Denisenko & Karol Bomsztyk, Nature Protocols 1, 179-185 (2006)]. Commercial companies, such as Roche's SeqCap EZ Human Exome Library v3.0 and Agilent's SureSelect, also depend on microsphere and/or bead methods to enrich targets. There has been a long-felt unsolved need for an easier alternative method for target enrichment, especially in a multiplex format, which can be further applied to target purification if purity is also desired.
In the post-genomic era, life science research and clinical applications are moving towards pathway, biomarker based studies. Biomarkers are guiding the drug development process, and they are crucial in the formation of the best treatment plans for patients or personalized medicine [“Integration and use of biomarkers in drug development, regulation and clinical practice: a US regulatory perspective,” Shashi Amur et al, Biomarkers Med. (2008) 2(3), 305-31]. A validated biomarker panel for clinical research or practice and routine studies usually contains less than 10 analytes [“Multitarget stool DNA testing for colorectal-cancer screening”, N Engl J Med. 2014 Apr. 3; 370 (14):1287-97]. Ideally, biomarkers can be measured via non-invasive methods, which require them to be present in peripheral body tissue and/or fluid, such as blood, urine, etc. Also, the methods to detect or measure the biomarkers need to be easy, fast, affordable, reproducible and robust [“Biomarkers on a roll”, Nature Biotechnology 28, 431 (2010)]. Few of the aforementioned multiplex assays meet these criteria.
There is a great demand for a user friendly, flexible, reproducible, affordable, low density and routine multiplex assay system to capitalize on genomic information in bettering our lives. Therefore, there is a need in the art to develop simplified methods for multiplex assays and multiplex target enrichment or purification so as to make them a routine lab tool in research and clinical practice. The present invention satisfies these long-felt, unsolved needs.